Immunotherapy infusing antibodies, vaccines, and cells is an effective treatment approach for human malignancies. Potent anti-tumor effects are realized by harnessing the specificity and associated effector functions of the immune system to recognize and eliminate cancer cells. Clinical trials have revealed the advantages of immune-based therapies, including (i) defined mechanisms of action, (ii) defined specificity and reduced deleterious off-target effects, (iii) lower toxicities than conventional approaches. Adoptive cell therapy (ACT), based on the adoptive transfer of T cells genetically modified to enforce expression of a chimeric antigen receptor (CAR), has shown considerable promise in clinical trials treating tumors refractory to all other treatment methods. In particular, the use of CAR+ T cells rendered specific for CD19 demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL) refractory to conventional therapies. These trials infuse a heterogeneous population of genetically modified T cells which have been propagated to clinically-sufficient numbers. While the therapeutic potential of infused T cells depends on their persistence, immunocorrelative studies evaluating survival of infused T cells are currently limited to (i) describing the immunophenotypes and function of whole populations of the T-cell inoculum and (ii) immunophenotypic analyses on T cells recovered after infusion. This grant application seeks to adapt single- cell imaging and retrieval to inform on the potency of clinical-grade CD19-specific CAR+ T cells by developing a unified platform for assessing both phenotype and function on the few T cells (and tumor cells) directly obtained (without in vitro manipulation) from recipients of immunotherapy. Our objective is to use high throughput single-cell nanowell screening (SNS) that we have developed to undertake an in-depth quantitative functional characterization (multiplexed cytokine secretion, phenotype, cytotoxicity, effect of regulatory T cells, Treg) of pre-infusion CAR+ T cells and compare these data to T cells recovered from the patient, post-infusion. These data will be used to (i) quantify the functionalit of infused T cells and the potential for anti-tumor effects and (ii) improve the generation of T cells for greater efficacy in next-generation clinical trials. We will validate our approach in Specific Aim 1 where we will define the integrated functional and molecular profiles of clinical- grade CD19-specific CAR+ T cells. In Specific Aim 2 we will implement our methodology to quantify the in vivo persistence of adoptively transferred cells. This will test the hypothesis tha SNS can quantify the therapeutic potential of clinical-grade T cells and in the formation of next-generation clinical trials.